Stem cells retain the ability to renew themselves through mitotic cell division and can differentiate into a diverse range of specialized cell types. Therefore they are considered potentially useful for treatment of a large variety of human and animal conditions. A number of adult stem cell therapies already exist, particularly bone marrow transplants that are used to treat leukaemia and in the future stem cell therapies are anticipated to enable the treatment cancer, Parkinson's disease, spinal cord injuries but also the replacement and repair of tissues such as pancreatic islets, severed nerve cells, skin grafts for burns or abrasions, and haematopoietic cells following chemotherapy and radiation.
Mesenchymal stem and progenitor cells are of great therapeutic potential due to their capacity of self-renewal and multilineage differentiation. They support haematopoiesis and enhance the engraftment of haematopoietic stem cells after co-transplantation. Currently, bone marrow represents the major source of mesenchymal stem and progenitor cells for cell therapy. However, aspiration of bone marrow involves invasive procedures and the frequency and differentiation potential of bone marrow derived mesenchymal stem and progenitor cells decrease significantly with age. Furthermore, the use of bone marrow is not always acceptable due to higher risk for viral infection and the lower number of cells that can be obtained from bone marrow.
Adipose tissue or fat tissue represents an accessible source of stem and progenitor cells, with similar characteristics to bone marrow-derived stem cells. Therefore adipose tissue may be an ideal source of high amounts of autologous stem cells. Adipose tissue derived mesenchymal stem and progenitor cells can for instance be obtained from adipose tissue obtained during liposuction surgery. Since liposuction aspirates are considered as a waste product these liposuction aspirates are a possible source for acquiring adipose tissue derived mesenchymal stem and progenitor cells.
It remains however necessary to preserve these adipose tissues for prolonged periods and cryopreservation is a method for doing such. Cryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as −196° C. At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. However, appropriate tissue-specific cryoprotection media are required in order to preserve the tissue or cells without causing freezing damage during the approach to low temperatures or warming to room temperature.
The patent application WO03024215 discloses how stem and progenitor cells derived from adipose tissue can be cryopreserved. The proposed method requires the isolation of the stem and progenitor cells from the adipose tissue prior to the cryopreservation. However, these steps are often unnecessary since only a small amount of the cryopreserved samples are actually being used.
Therefore there is a need for a method for cryopreserving adipose tissue under current good manufacturing practices and current good tissue practices, and under conditions that do not affect the biological characteristics of the tissue. When necessary the cryopreserved adipose tissue can subsequently be used for isolating stem and progenitor cells with the intention to use the cells for therapeutic purposes.
Attempts to cryopreserve adipose tissue and separate the stem and progenitor cells after thawing have until now not produced useful amounts of viable stem and progenitor cells.
Furthermore, some methods for cryopreservation and isolation of stem and/or progenitor cells use xenogeneic sera as cryoprotectant or in the cell culture media. These xenogeneic sera, like fetal bovine serum, are a potential source of contaminants, including prions, viruses and mycoplasms, and should therefore be avoided. Furthermore, the high endotoxin content of for instance fetal bovine serum also poses a safety risk. The presence of xenogeneic sera in the culture or cryopreservation of cells have also been found to change expression patterns of genes and induce unstable transcriptional profiles that for instance lead to an overexpression of collagen, changing the adherence characteristics of the cells. Thus cells contacted with a xenogeneic serum or plasma can display significantly different cell expression profiles from cells prior to the contact, and are substantially altered physiologically, functionally and even genetically as a result of the contact with xenogeneic materials.
It is the intention of the present invention to provide a method for the cryopreservation of adipose tissue. The cryopreservation method of the present invention provides a method for the subsequent isolation of a large amount of viable stem and/or progenitor cells from the cryopreserved adipose tissue. The obtained stem and/or progenitor cells can be used for clinical therapies as well as for drug screening or for developing procedures for expansion or differentiation.